Translation of dipeptide repeat proteins in C9ORF72 ALS/FTD through unique and redundant AUG initiation codons
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This valuable study by Sonobe et al uses transfected cells and patient iPSC-derived neurons to define mechanisms underlying translation of the antisense C4G2 RNA strand expressed in C9orf72-associated ALS and FTD. The authors design a series of constructs to explore the start codon required to produce toxic PR and prominent PG dipeptides in disease. Using these constructs they provide solid data that translation in the PR and PG reading frames occur due to the presence of AUG codons within the 5'UTR of the RNA strand. However, in its current form the paper is incomplete and the conclusions require additional experimental support.
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Abstract
A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A hallmark of ALS/FTD pathology is the presence of dipeptide repeat (DPR) proteins, produced from both sense GGGGCC (poly-GA, poly-GP, poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) transcripts. Translation of sense DPRs, such as poly-GA and poly-GR, depends on non-canonical (non-AUG) initiation codons. Here, we provide evidence for canonical AUG-dependent translation of two antisense DPRs, poly-PR and poly-PG. A single AUG is required for synthesis of poly-PR, one of the most toxic DPRs. Unexpectedly, we found redundancy between three AUG codons necessary for poly-PG translation. Further, the eukaryotic translation initiation factor 2D (EIF2D), which was previously implicated in sense DPR synthesis, is not required for AUG-dependent poly-PR or poly-PG translation, suggesting that distinct translation initiation factors control DPR synthesis from sense and antisense transcripts. Our findings on DPR synthesis from the C9ORF72 locus may be broadly applicable to many other nucleotide repeat expansion disorders.
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Author Response
Reviewer #2 (Public Review):
Dipeptide repeat (DPR) proteins produced from both sense GGGGCC (poly-GA, poly-GP and poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) repeat RNAs are found C9ORF72-linked ALS/FTD and contribute to neurodegeneration. The translation of the repeat RNA can initiate without the AUG start codon, a process known as repeat associated non-AUG (RAN) translation. In this manuscript, the authors used luciferase reporter construct to show that the translation of PR and PG from the CCCCGG repeats initiated from in-frame AUG in the C9 sequences before the repeats. After mutating candidate AUG codons, the translation can initiate from other AUG, so there is redundancy. But if mutating all the in-frame AUG codons, the luciferase was dramatically reduced, supporting the translation initiated at …
Author Response
Reviewer #2 (Public Review):
Dipeptide repeat (DPR) proteins produced from both sense GGGGCC (poly-GA, poly-GP and poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) repeat RNAs are found C9ORF72-linked ALS/FTD and contribute to neurodegeneration. The translation of the repeat RNA can initiate without the AUG start codon, a process known as repeat associated non-AUG (RAN) translation. In this manuscript, the authors used luciferase reporter construct to show that the translation of PR and PG from the CCCCGG repeats initiated from in-frame AUG in the C9 sequences before the repeats. After mutating candidate AUG codons, the translation can initiate from other AUG, so there is redundancy. But if mutating all the in-frame AUG codons, the luciferase was dramatically reduced, supporting the translation initiated at the AUG start codon. The translation initiation factor eIF2D has been shown to be important for CUG start codon-dependent poly-GA translation from GGGGCC repeats. Here it is shown that eIF2D is not required for poly-PG and poly-PR translation from CCCCGG repeats using both reporter and patient iPS-neurons. The data using luciferase reporter to study antisense repeat translation is solid, the translation initiates from AUG start codon as there are AUG in frame with PG and PR in the constructs containing the antisense sequences.
We thank the reviewer for the constructive feedback.
On the other hand, as the reporter construct includes the sequences containing the AUG codon, it is not surprising that AUG was used. This is canonical translation.
We completely agree. In the revised Introduction, we now point out that, before our study, it was not clear which mode of translation (RAN vs AUG canonical) is employed for DPR synthesis.
Also, in the revised Discussion (lines 251-257)), we mention the following: “Hence, our findings together with these previous studies suggest that DPR synthesis may involve at least three different modes of translation: (a) near-cognate start codon (e.g., CUG, AGG) dependent-translation for poly-GA and poly-GR from sense GGGGCC transcripts, (b) canonical AUG-dependent translation for poly-PR and poly-PG synthesis from antisense CCCCGG transcripts, and (c) DPR synthesis may also occur through RAN translation mechanisms that solely utilize the repeat. It is conceivable that all three modes of translation may occur simultaneously in disease, and that the use of non-canonical and canonical initiation codons may be the primary contributors of DPR production ”.
The 1,000bp intronic sequence included in our antisense 35xCCCCGG constructs (Figure 1A) is the authentic human intronic sequence. We agree that it does contain multiple putative initiation codons, and this was our motivation for conducting systematic mutagenesis of all these codons. To narrow down the list of putative initiation codons, we used our recently developed machine-learning algorithm for initiation codon prediction (PMID: 35648796). We found a CUG and an AUG in poly-PR frame; a CUG and three AUGs in the poly-PG frame), all of which had a good Kozak sequence (as mentioned in Results). Systematic mutagenesis of these codons (single and multiple codon mutations were generated) revealed that an AUG at -273bp is necessary for poly-PR synthesis (Figure 2). Of note, poly-PR is one of the most toxic DPRs, for which an initiation codon had not been previously identified in the literature.
Additionally, the AUG-initiated translation of antisense repeats has been reported previously. Therefore, the novelty is limited.
We agree that an AUG initiation codon was previously described for poly-PG (Boivin et al., EMBO J, 2020, PMID: 31930538). However, our findings significantly extend this observation because redundancy at the level of AUG initiation codon usage was not reported in that study.
We believe our study significantly contributes to the field of C9ORF72 ALS/FTD in the following way:
(i) We identified for the first time an AUG (at -273nt) necessary for synthesis of poly-PR, one of the most toxic DPRs.
(ii) We propose the concept of initiation codon redundancy for poly-PG, which may apply to other DPRs in C9ORF72 ALS/FTD, as well as in other neurological disorders caused by nucleotide repeat expansion mutations.
(iii) Our findings merged with those of previous studies suggest that DPR synthesis may involve at least three different modes of translation: (a) near-cognate start codon (e.g., CUG, AGG) dependent-translation for poly-GA and poly-GR from sense GGGGCC transcripts, (b) canonical AUG-dependent translation for poly-PR and poly-PG synthesis from antisense CCCCGG transcripts, and (c) DPR synthesis may also occur through RAN translation mechanisms that solely utilize the repeat. It is conceivable that all three modes of translation may occur simultaneously in disease, and the use of non-canonical and canonical initiation codons may be the primary contributor of DPR production”.
(iv) We found that the non-canonical translation initiation factor eIF2D is mainly responsible for poly-GA (sense DPR) production without affecting anti-sense DPRs. Hence, we propose a model where DPR translation occurs in a “piecemeal manner”, i.e., a distinct machinery of translation initiation factors may be needed for the synthesis of each DPR.
In the revised manuscript, we now better highlight these key contributions.
How the antisense DPRs are translated endogenously, AUG-canonical translation or RAN translation, depends on whether the AUG is included in the antisense RNA in patients and where the transcription of the antisense starts, upstream or downstream of the AUG start codons. However, this is not considered in the manuscript.
Thank you for this important point. Zu et al., (PNAS, 2013) observed antisense DPR aggregation in brain samples of C9ORF72 ALS/FTD patients. In the same study, the authors conducted 5’ Rapid Amplification of cDNA Ends (RACE). Although this analysis did not identify the exact transcription start site for the antisense CCCCGG RNA, it did show that the region that includes the AUG codons, which we found to be important for poly-PR or poly-PG, is included in the antisense RNA from human C9ORF72 ALS/FTD samples. In page E4969, Zu et al write: “RACE analysis of FCX samples showed intron 1b antisense transcripts begin at varying sites 251–455 bp upstream of the G2C4 repeat”. The same study also detected antisense RNA foci in brain samples of C9ORF72 ALS/FTD patients.
The exact transcription start site for the antisense (and sense) transcript remains unknown. In the near future, we plan RACE experiments to identify it and share these finding with the community in a separate manuscript.
We have modified the Results (lines 133-136) to: “These results strongly suggest that AUG at -273 bp is the start codon for translation of poly-PR, one of the most toxic DPRs in C9ORF72 ALS/FTD. This AUG is predicted to be included in the endogenous antisense CCCCGG transcript based on 5’ Rapid Amplification of cDNA Ends (RACE) analysis on brain samples of C9ORF72 ALS/FTD patients14.”
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eLife assessment
This valuable study by Sonobe et al uses transfected cells and patient iPSC-derived neurons to define mechanisms underlying translation of the antisense C4G2 RNA strand expressed in C9orf72-associated ALS and FTD. The authors design a series of constructs to explore the start codon required to produce toxic PR and prominent PG dipeptides in disease. Using these constructs they provide solid data that translation in the PR and PG reading frames occur due to the presence of AUG codons within the 5'UTR of the RNA strand. However, in its current form the paper is incomplete and the conclusions require additional experimental support.
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Reviewer #1 (Public Review):
Sonobe et al provide compelling data on the translation initiation codons required for PR and PG production from the antisense C4G2 repeat expansion associated with C9orf72-ALS/FTD. The strengths of this study are the systematic approach by which the authors identify the start codons. They also provide data investigating if eIF2D is needed for DPR production, building upon previous findings. A major weakness of this work includes the lack of full characterization of their models as they relate to disease, including the need to define any potential toxicity associated with DPR production and any DPR aggregation. Additionally, the study would be strengthened by the quantification and further validation of some of the data presented. Overall, the findings within the study have the potential to be important in …
Reviewer #1 (Public Review):
Sonobe et al provide compelling data on the translation initiation codons required for PR and PG production from the antisense C4G2 repeat expansion associated with C9orf72-ALS/FTD. The strengths of this study are the systematic approach by which the authors identify the start codons. They also provide data investigating if eIF2D is needed for DPR production, building upon previous findings. A major weakness of this work includes the lack of full characterization of their models as they relate to disease, including the need to define any potential toxicity associated with DPR production and any DPR aggregation. Additionally, the study would be strengthened by the quantification and further validation of some of the data presented. Overall, the findings within the study have the potential to be important in advancing our understandings of toxic dipeptide production from the G4C2 repeat expansion in C9orf72-ALS/FTD. This has implications clinically and increases our biological interpretation of this disease.
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Reviewer #2 (Public Review):
Dipeptide repeat (DPR) proteins produced from both sense GGGGCC (poly-GA, poly-GP and poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) repeat RNAs are found C9ORF72-linked ALS/FTD and contribute to neurodegeneration. The translation of the repeat RNA can initiate without the AUG start codon, a process known as repeat associated non-AUG (RAN) translation. In this manuscript, the authors used luciferase reporter construct to show that the translation of PR and PG from the CCCCGG repeats initiated from in-frame AUG in the C9 sequences before the repeats. After mutating candidate AUG codons, the translation can initiate from other AUG, so there is redundancy. But if mutating all the in-frame AUG codons, the luciferase was dramatically reduced, supporting the translation initiated at the AUG start codon. …
Reviewer #2 (Public Review):
Dipeptide repeat (DPR) proteins produced from both sense GGGGCC (poly-GA, poly-GP and poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) repeat RNAs are found C9ORF72-linked ALS/FTD and contribute to neurodegeneration. The translation of the repeat RNA can initiate without the AUG start codon, a process known as repeat associated non-AUG (RAN) translation. In this manuscript, the authors used luciferase reporter construct to show that the translation of PR and PG from the CCCCGG repeats initiated from in-frame AUG in the C9 sequences before the repeats. After mutating candidate AUG codons, the translation can initiate from other AUG, so there is redundancy. But if mutating all the in-frame AUG codons, the luciferase was dramatically reduced, supporting the translation initiated at the AUG start codon. The translation initiation factor eIF2D has been shown to be important for CUG start codon-dependent poly-GA translation from GGGGCC repeats. Here it is shown that eIF2D is not required for poly-PG and poly-PR translation from CCCCGG repeats using both reporter and patient iPS-neurons. The data using luciferase reporter to study antisense repeat translation is solid, the translation initiates from AUG start codon as there are AUG in frame with PG and PR in the constructs containing the antisense sequences.
On the other hand, as the reporter construct includes the sequences containing the AUG codon, it is not surprising that AUG was used. This is canonical translation. Additionally, the AUG-initiated translation of antisense repeats has been reported previously. Therefore, the novelty is limited. How the antisense DPRs are translated endogenously, AUG-canonical translation or RAN translation, depends on whether the AUG is included in the antisense RNA in patients and where the transcription of the antisense starts, upstream or downstream of the AUG start codons. However, this is not considered in the manuscript.
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